Reagents and methods for autoligation chain reaction

ABSTRACT

The invention relates to the amplification of specific target nucleic acids. The invention provides methods, reagents, and kits for carrying out such amplification via the autoligation chain reaction (ACR).

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No.14/370,446, filed Jul. 2, 2014, now U.S. Pat. No. 9,725,761, issued Aug.8, 2017, which is a National Stage Entry of International ApplicationNo. PCT/US2012/072192, filed Dec. 28, 2012, which claims the benefit ofU.S. Provisional Patent Application No. 61/580,988, filed Dec. 28, 2011,each of which is entirely incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under SBIR Phase I AwardIIP-1046508 and SBIR Phase II Award IIP-1230464, both awarded by theNational Science Foundation. The government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 8, 2013, isnamed 44086-701.601_SL.txt and is 4,714 bytes in size.

BACKGROUND OF THE INVENTION

Amplification of nucleic acid sequences is a widespread technology thathas been used for many purposes, including diagnostic and forensictesting. Currently, this is carried out using polymerase chain reaction(PCR). Unfortunately, critical barriers exist with PCR that prevent bothclinical and research labs from adopting PCR-based assays into a routinesetting, due to bottlenecks with sample preparation and assaydevelopment costs. Specifically, PCR inhibitors, such as inhibitors topolymerases, found in many laboratory samples and clinical specimenscause low sensitivity and false-negative results in clinical andforensic tests that rely on PCR-based molecular techniques. Therefore,it is widely accepted that purification or pre-amplification of targetDNA nucleic acids is required to remove or dilute out inhibitors priorto PCR amplification to obtain successful results. Optimization of PCRfor genetic testing with different sample types can be labor-intensive,requiring extensive amounts of upfront development work, which in turncan significantly increase both the overall cost of a test and thetime-to-result. See Al-Soud, W. A. & Rådström, P. (2001). Purificationand Characterization of PCR-Inhibitory Components in Blood Cells.Journal of Clinical Microbiology, 39(2), 485-493; Huggett, J. F., Novak,T., Garson, J. A., Green, C., Morris-Jones, S. D., Miller, R. F. &Zumla, A. (2008). Differential susceptibility of PCR reactions toinhibitors: an important and unrecognised phenomenon. BMC ResearchNotes, 1(70), 1-9; Ochert, A. S., Boulter, A. W., Birnbaum, W., Johnson,N. W. & Teo, C. G. (1994). Inhibitory effect of salivary fluids on PCR:potency and removal. Genome Res., 3, 365-368; Ratnamohana, V. M.,Cunningham, A. L., & Rawlinson, W. D. (1998). Removal of inhibitors ofCSF-PCR to improve diagnosis of herpesviral encephalitis. Journal ofVirological Methods, 72(1), 59-65; and Honoré-Bouakline, S., Vincensini,J. P., Giacuzzo, V., Lagrange, P. H. & Herrmann, J. L. (2003). RapidDiagnosis of Extrapulmonary Tuberculosis by PCR: Impact of SamplePreparation and DNA Extraction. Journal of Clinical Microbiology, 41(6),2323-2329.

With the upsurge in genetic information and the resultant increase inDNA biomarkers, researchers are now seeking new technologies to rapidlyand cost-effectively interrogate this new information in a routinesetting. However, the critical barriers associated with PCR make thistechnology too cost-prohibitive and too labor-intensive to use as atesting method for price-sensitive laboratories with limited resourcesand large numbers of samples.

Recently, technology has been developed to detect and monitor cellulargenetic mutations using RNA-templated chemistry without amplification ofthe RNA template, in which chemically modified probes fluoresce whenthey hybridize to their genetic target in intact bacterial and humancells. See Franzini, R. M. and Kool, E. (2008). 7-Azidomethoxy-coumarinsas profluorophores for template nucleic acid detection. Chem Bio Chem 9:2981-2988; Franzini, R. M. and Kool, E. (2009). Efficient nucleic aciddetection by template reductive quencher release. J. Am. Chem. Soc. 131:16021-16023; Silverman, A. P. and Kool, E. (2005). Quenched autoligationprobes allow discrimination of live bacterial species by singlenucleotide differences in rRNA. Nucleic Acids Res. 33: 4978-4986; Sando,S. and Kool, E. (2002). Nonenzymatic DNA ligation in Escherichia colicells. Nucleic Acids Res. Supplement No. 2: 121-122; Abe, H. and Kool.,E. (2006). Flow cytometric detection of specific RNAs in native humancells with quenched autoligating FRET probes. Proc. Natl. Acad. Sci. USA103: 263-268; Sengen Sun and Joseph A. Piccirilli. (2010). Synthesis of3′-Thioribouridine, 3′-Thioribocytidine, and Their Phosphoramidites.Nucleosides, Nucleotides and Nucleic Acids. 16(7): 1543-1545.

This probe-based strategy, called quenched autoligation (“QUAL”),utilizes two self-reacting oligonucleotide probes that provide afluorescence signal in the presence of fully complementary nucleic acidtarget sequence. A first oligonucleotide having a3′-phosphoromono-thioate nucleophilic group anneals to a template targetsequence, such that the 3′-phosphoromono-thioate nucleophilic group isjuxtaposed to a 5′-electrophilic dabsylated group quencher of a secondannealed oligonucleotide which has a fluorescein group quenched by thedabsyl group. This tandem configuration along a DNA template catalyzesthe autoligation reaction, and joins the two oligonucleotides into asingle probe. Upon ligation, the dabsyl quencher is displaced, and thefluoresceinyl fluorophore becomes un-quenched, resulting in an increasein fluorescence signal.

These short QUAL probes have been used to distinguish closely relatedbacterial species by discriminating single nucleotide differences in 16SrRNA sequences within live cells. However, QUAL is not compatible within vitro applications that require the detection of small amounts ofdouble-stranded nucleic acid sequences that are typically found insamples used for routine genetic testing of DNA biomarkers. For example,a QUAL in vitro reaction typically contains 10¹³ copies ofsingle-stranded oligo DNA template, but a routine molecular assay cancontain 10³ or fewer copies of dsDNA biomarkers—a ten billion-folddifference in copy-number detection. QUAL does not provide a way toamplify the signal resulting from the ligation reaction, because theproduct of the ligation reaction is a nucleic acid which is stablyannealed to the template, thus occupying the template and not permittingit to participate in additional signal-generating reactions. Denaturingthe reaction product from the template requires high temperatures atwhich the QUAL probes would be degraded. The autoligation chemistriesused in QUAL have reduced stability at the high temperatures needed toseparate double-stranded DNA.

There is, therefore, a need for methods, reagents, and kits foramplifying nucleic acid sequences without enzymes or nucleosides toenable cost-effective and easier-to-use alternatives for genetic testingthat can be implemented in routine settings across multiple sample typeswithout any sample-prep development.

BRIEF SUMMARY OF THE INVENTION

The invention relates to amplification of nucleic acid sequences. Moreparticularly, the invention relates to amplification of nucleic acidsequences without enzymes or nucleosides. The invention providesreagents and methods for amplifying nucleic acid sequences withoutenzymes or nucleosides and effective at temperatures which denaturenucleic acids.

The invention provides a method for amplifying a specific target nucleicacid sequence. In some embodiments, the invention provides a method forlinearly amplifying a specific target nucleic acid sequence. In someembodiments, the invention provides a method for exponentiallyamplifying a specific target nucleic acid sequence. The method accordingto this aspect of the invention comprises contacting the target nucleicacid sequence with a first forward primer nucleic acid, a second forwardprimer nucleic acid, a first reverse primer nucleic acid and a secondreverse primer nucleic acid under conditions wherein the primer nucleicacids specifically anneal with the target nucleic acid sequence. Oneforward primer nucleic acid has a first bond-forming reactive moiety andthe other forward primer nucleic acid has a second bond-forming reactivemoiety. One reverse primer nucleic acid has a first bond-formingreactive moiety and the other reverse primer nucleic acid has a secondbond-forming reactive moiety. The first forward primer nucleic acid andthe second forward primer nucleic acid are annealed to the targetnucleic acid sequence such that the reactive moiety of the first forwardprimer nucleic acid and the reactive moiety of the second forward primernucleic acid are juxtaposed. The first reverse primer nucleic acid andthe second reverse primer nucleic acid are annealed to the targetnucleic acid sequence such that the reactive moiety of the first reverseprimer nucleic acid and the reactive moiety of the second reverse primernucleic acid are juxtaposed. The reactive moiety of the first forwardprimer nucleic acid forms a chemical bond with the reactive moiety ofthe second forward primer nucleic acid to form a first ligation product,and the reactive moiety of the first reverse primer nucleic acid forms achemical bond with the reactive moiety of the second reverse primernucleic acid to form a second ligation product. Thus, the first ligationproduct forms a duplex with the target nucleic acid sequence and thesecond ligation product forms a duplex with the target nucleic acidsequence. The duplexes are then disrupted to form target nucleic acidsequences and the steps are repeated to amplify the target nucleic acidsequences. In some embodiments, the duplexes are thermally disrupted toform target nucleic acid sequences and the steps are repeated to amplifythe target nucleic acid sequences.

In some embodiments, the ligation product formed by ligation of theforward or reverse primers does not comprise more than 1, 2, 3, 4, 5, or6 bases which are not paired with the target nucleic acid sequence. Forexample, the ligation product formed by ligation of the forward orreverse primers does not comprise more than 2 bases which are not pairedwith the target nucleic acid sequence.

In some embodiments, a cycle consisting of steps (a) and (b) isperformed in less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes. In someembodiments, the target nucleic acid sequence is present in the samplein a low copy number. For example, the sample comprises less than about10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², or 10 copies.

In some embodiments, the first bond-forming reactive moiety is an azideand the second bond-forming reactive moiety is an alkyne. In someembodiments, the first bond-forming reactive moiety is an alkyne and thesecond bond-forming reactive moiety is an azide. In some embodiments,the target nucleic acid sequence is amplified exponentially. In otherembodiments, the target nucleic acid sequence is amplified linearly.

In some embodiments, one forward or reverse primer nucleic acidcomprises a dye or detectable group. In some embodiments, one forward orreverse primer nucleic acid comprises a fluorescence resonance energytransfer (FRET) donor fluorophore and/or the other forward or reverseprimer nucleic acid comprises a FRET acceptor fluorophore, and theligation products are detected by FRET. In some embodiments, the dye ordetectable group is quenched by a quenching moiety in which annealingand autoligation separates the quenching moiety from the dye ordetectable group before the ligated product is detected.

In some embodiments the forward and reverse primer nucleic acids containneither a dye nor a detectable group, and the ligation products aredetected by double-stranded nucleic acid binding dyes.

The invention further provides reagent compositions for amplifying atarget nucleic acid sequence. In some embodiments, the inventionprovides reagent compositions for linearly amplifying a specific targetnucleic acid sequence. In some embodiments, the invention providesreagent compositions for exponentially amplifying a specific targetnucleic acid sequence. In some embodiments, a reagent compositionaccording to the invention comprises a first forward primer nucleic acidhaving a thermally stable first bond-forming reactive moiety. In someembodiments, a reagent composition according to the invention comprisesa second forward primer nucleic acid having a thermally stable secondbond-forming reactive moiety. In some embodiments, a reagent compositionaccording to the invention comprises a first reverse primer nucleic acidhaving a thermally stable first bond-forming reactive moiety. In someembodiments, a reagent composition according to the invention comprisesa second reverse primer nucleic acid having a thermally stable secondbond-forming reactive moiety. In such embodiments, the firstbond-forming reactive moiety forms a chemical bond with the secondbond-forming reactive moiety, when the first forward primer nucleic acidand the second forward primer nucleic acid are juxtaposed by annealingwith a target nucleic acid and when the first reverse primer and thesecond reverse primer nucleic acid are juxtaposed by annealing with atarget nucleic acid. In some embodiments, the first bond-formingreactive moiety is an alkyne (for example a hexynyl or octadiynyl group)and the second bond-forming reactive moiety is an azide. In someembodiments, the first bond-forming reactive moiety is an azide and thesecond bond-forming reactive moiety is an alkyne (for example a hexynylor octadiynyl group). In some embodiments, one forward or reverse primernucleic acid comprises a dye or detectable group. In some embodiments,one forward or reverse primer nucleic acid comprises a FRET donorfluorophore and/or the other forward or reverse primer nucleic acidcomprises a FRET acceptor fluorophore. In some embodiments, the dye ordetectable group is quenched by a quenching moiety in which annealingand autoligation separates the quenching moiety from the dye ordetectable group before the ligated product is detected. In someembodiments the forward and reverse primer nucleic acids contain neithera dye nor a detectable group, and the ligation products are detected bydouble-stranded nucleic acid binding dyes.

Also provided are reaction mixtures comprising a reagent composition asdescribed herein. In some embodiments, reaction mixtures comprise targetnucleic acid sequences, including single-stranded and double-strandedtarget nucleic acid sequences. Reaction mixtures of the invention mayfurther comprise any needed reagents, including buffers, salts, or dyes.

Provided herein is a kit for amplifying a target nucleic acid sequence.In some embodiments, the invention provides a kit for linearlyamplifying a specific target nucleic acid sequence. In some embodiments,the invention provides a kit for exponentially amplifying a specifictarget nucleic acid sequence. The kit according to this aspect of theinvention comprises a first forward primer nucleic acid, a secondforward primer nucleic acid, a first reverse primer nucleic acid, and asecond reverse primer nucleic acid. In the kit according to this aspectof the invention, the first forward primer nucleic acid, the secondforward primer nucleic acid, the first reverse primer nucleic acid, andthe second reverse primer nucleic acid are as described for the secondaspect according to the invention. In some embodiments, the kit furthercomprises a second reagent composition as described herein, wherein thesecond reagent composition is designed for the amplification of at leasta second target nucleic acid sequence. In some embodiments, the at leasta second target nucleic acid sequence differs from the target nucleicacid sequence by at least a single nucleotide or nucleotide base pair,for example 1, 2, 3, 4 or more nucleotides or nucleotide base pairs.

Provided herein is also a method of detecting amplification of a nucleicacid target comprising: (a) contacting the target nucleic acid sequencewith a first forward primer nucleic acid, a second forward primernucleic acid, a first reverse primer nucleic acid and second reverseprimer nucleic acid under conditions wherein the first and secondforward primer nucleic acids specifically anneal with the target nucleicacid sequence and are juxtaposed on the target nucleic acid sequence;the first and second forward primer nucleic acids covalently ligate toeach other upon binding to the target nucleic acid sequence, resultingin a first ligation product which forms a first duplex with the targetnucleic acid sequence; the first and second reverse primer nucleic acidscovalently ligate to each other upon binding to the target nucleic acidsequence, resulting in a first ligation product which forms a secondduplex with the target nucleic acid sequence; (b) disrupting the duplexto release template target nucleic acid sequences and repeating step(a); and (c) during steps (a) or (b), detecting a change in a detectablesignal, wherein the change is proportional to the amount of ligationproducts in the sample. For example, the signal is a fluorescent signal.In some embodiments, step (c) comprises determining an absolute orrelative amount of target nucleic acid sequence. In some embodiments,the amplification is exponential. In some embodiments, the method isused in the amplification of at least a second target nucleic acidsequence, for example wherein the second target nucleic acid sequencediffers from the target nucleic acid sequence by at least a singlenucleotide or nucleotide base pair, for example by 1, 2, 3, 4 or morenucleotides or nucleotide base pairs.

Provided herein is a device for performing nucleic acid amplification ofa target nucleic acid sequence, comprising: (a) an automated thermalcycler capable of alternately heating and cooling at least one reactionvessel comprising the reagent composition of the invention; (b) anexcitation source for optically exciting the sample and causing thesample to fluoresce; and (c) a photodetector for detecting a fluorescentsignal from the sample while the amplification reaction is in progress,which fluorescent signal is proportional to the amount of amplifiednucleic acid in the reaction vessel.

Further provided is a method performing nucleic acid amplification of afirst target nucleic acid sequence comprising: (a) mixing, in at leastone reaction vessel, a dsDNA binding dye with a sample comprising areagent composition of the invention and the first target nucleic acidsequence; (b) amplifying the first target nucleic acid sequence byalternately heating and cooling the reaction vessel; (c) detecting thefluorescence of the dsDNA binding dye by melting amplified targetnucleic acid to generate a first melting curve; (d) repeating themixing, amplifying and detecting steps with a second target nucleic acidsequence to generate a second melting curve; and (e) comparing the firstand second melting curves to determine a difference in the nucleic acidcomposition of the first and second target nucleic acid sequences. Insome embodiments, the difference is a single nucleotide or nucleotidebase pair.

The invention further provides a device for performing nucleic acidamplification of a target nucleic acid sequence, comprising: (a) atleast one reaction vessel comprising the reagent composition of theinvention; (b) an excitation source for optically exciting the sampleand causing the sample to fluoresce; (c) a photodetector for detectingtemperature-dependent fluorescence levels from the sample; and (d) aprocessor programmed to generate a melting curve of the amplificationproduct contained within the reaction vessel. For example, the device isconfigured to alternately heat and cool the reaction vessel.

Also provided is a method of amplifying of a target nucleic acidsequence comprising: (a) contacting the target nucleic acid sequencewith a first forward primer nucleic acid and a second forward primernucleic acid, under conditions wherein the first and second forwardprimer nucleic acids specifically anneal with the target nucleic acidsequence and are juxtaposed on the target nucleic acid sequence, whereinthe first and second forward primer nucleic acids covalently bond uponbinding to the target nucleic acid sequence, resulting in a ligationproduct which forms a duplex with the target nucleic acid sequence; and(b) disrupting the duplex to release the template target nucleic acidsequences and repeating step (a). In some embodiments, a cycleconsisting of steps (a) and (b) is performed in less than 2, 3, 4, 5, 6,7, 8, 9 or 10 minutes.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the general strategy and expected results from tworounds of Autoligation Chain Reaction (“ACR”), in which fourdouble-stranded products are generated from the amplification of asingle nucleic acid target sequence. Various bond-forming reactivemoieties (BFRM) can be used with ACR.

FIG. 2 illustrates the strategy and expected results from two rounds ofACR, in which four double-stranded products are generated from theamplification of a single nucleic acid target sequence, and detectionwith double-stranded nucleic acid binding dyes (DBD). Different dyes canbe used with ACR.

FIG. 3 shows ACR self-ligation reactions with dsDNA nucleic acidtemplate using duplexed tandem primers or single tandem primers and SYBRGreen I for detection of binding to the duplexes formed fromamplification reactions in the presence or absence of a singlenucleotide polymorphism (SNP) mutation. This illustrates that ACR hasthe specificity to detect SNPs.

FIG. 4 illustrates the strategy and expected results from two rounds ofACR, in which four double-stranded products are generated from theamplification of a single nucleic acid target sequence, and detectionwith a detection group (Fluor) in which a quenching moiety (Quen) isseparated from the detection group after annealing and autoligationseparates the quenching moiety from the detectable group before theligated product is detected. This illustrates that different detectiongroups can be used with ACR.

FIG. 5 illustrates the strategy and expected results from two rounds ofACR, in which four double-stranded products are generated from theamplification of a single nucleic acid target sequence, and detectionwith detection groups (F1 and F2) by FRET. This illustrates thatdifferent detection strategies can be used with ACR.

FIG. 6 shows unstained and stained polyacrylamide gels after anautoligation reaction with a first forward primer nucleic acidcontaining a first bond-forming reactive moiety and a second forwardprimer nucleic acid containing a second bond-forming reactive moiety inwhich the second forward primer is labeled with FAM, thus illustratingthat different detection media and different detection platforms can beused with ACR.

FIG. 7 shows a stained polyacrylamide gel after an autoligation reactionwith a first reverse primer nucleic acid containing a first bond-formingreactive moiety and a second reverse primer nucleic acid containing asecond bond-forming reactive moiety in which the first and secondreverse primers are unlabeled.

FIG. 8 shows FAM/Texas Red FRET fluorescence of ACR reactions on anunstained 20% acrylamide+urea denaturing gel using the Typhoon Trio+imaging system.

FIG. 9 shows enrichment of ACR activity using different fractionselectrophilic ACR primers. Detection used FAM/Texas Red FRETfluorescence of ACR reactions on an unstained 20% acrylamide+ureadenaturing gel using the Typhoon Trio+ imaging system.

FIG. 10 shows enhancement of FRET using the same reactions with enrichedACR activity from FIG. 9 (Lanes 3 and 4) using two different channelsfor detection: Non-FRET and FRET channels.

FIG. 11A shows real-time amplification plots on a LightCycler® 480 IIinstrument using FAM/Texas Red FRET fluorescence in ACR reactions todemonstrate exponential amplification. FIG. 11B shows the same reactionproducts run on a SYBR Green I stained 20% acrylamide+urea denaturinggel using the Typhoon Trio+ imaging system. This illustrates that ACRcan be detected in real-time, and therefore ACR can be used for bothend-point and quantitative analyses.

FIG. 12 shows a non-limiting example of an ACR bond-forming chemistrythrough cycloaddition with alkyne and azide moieties to generate acovalent carbon-heteroatom bond between species to form a triazolconjugate.

FIG. 13 shows a non-limiting example of a bond-forming reactive moietyusing a hexynyl alkyne modification. This can be used to conjugate anACR Primer 2 to an ACR Primer 1 modified with an azide bond-formingreactive moiety.

FIG. 14 shows a non-limiting example of a bond-forming reactive moietyusing an octadiynyl alkyne modification. This can be used to conjugatean ACR Primer 2 to an ACR Primer 1 modified with an azide bond-formingreactive moiety.

FIG. 15 shows ACR primer sequences (SEQ ID NOS 5-16, respectively, inorder of appearance) containing an azide bond-forming reactive moiety(3AzideN),

and hexynyl (5Hexynyl) and octadiynyl (55 OCTdU) alkyne bond-formingreactive moieties to detect the maize Glutathione S-Transferase (GST)gene. Detection is through FRET using a fluorescein (iFluorT) and TAMRA(i6-TAMN) detection groups.

FIG. 16A shows real-time amplification plots of ACR with azide andhexynyl bond-forming reactive moieties detected on the LightCycler® 480II using FAM/TAMRA FRET fluorescence in ACR reactions to demonstrateexponential amplification down to 100 copies of template. FIG. 16B showsFRET ligation fluorescence of the same reaction products in lanes 1-3run on a 20% acrylamide+urea denaturing gel using the Typhoon Trio+imaging system. FRET ligation is not observed with lower concentrationsof the catalyst (lanes 4-9). This illustrates that azide and hexynylbond-forming reactive moieties have greater sensitivity with an enhancedexponential amplification profile over thiol nucleophilic andbromoacetate electrophilic moieties in ACR (FIGS. 11A and 11B), makingACR more amenable to both end-point and quantitative analyses.

FIG. 17 shows templated ligation of ACR reactions using azide andoctadiynyl bond-forming reactive moieties run on a 20% acrylamide+ureadenaturing gel using the Typhoon Trio+ imaging system. FRET templatedligation is observed down to 100 copies of template (lanes 2-7). Thisillustrates that azide and octadiynyl bond-forming reactive moietieshave greater sensitivity over thiol nucleophilic and bromoacetateelectrophilic moieties in ACR (FIGS. 11A and 11B), making ACR moreamenable to both end-point and quantitative analyses.

FIG. 18 shows thermal stability of bond-forming reactive moieties onfirst and second forward primer nucleic acids in which the secondforward primer nucleic acid contains FAM and FAM fluorescence of theautoligation reaction is detected on an unstained 20% acrylamide+ureadenaturing gel using the Typhoon Trio+ imaging system.

FIG. 19 shows FAM/Texas Red FRET fluorescence in ACR reactions withdecreasing amounts of template on an unstained 20% acrylamide+ureadenaturing gel using the Typhoon Trio+ imaging system.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to amplification of nucleic acid sequences. Moreparticularly, the invention relates to amplification of nucleic acidsequences without enzymes or nucleosides. The invention providesreagents, methods, kits and devices for amplifying nucleic acidsequences without enzymes or nucleosides.

In one aspect, the invention provides a method for amplifying a specifictarget nucleic acid sequence. In some embodiments, the inventionprovides a method for linearly amplifying a specific target nucleic acidsequence. In some embodiments, the invention provides a method forexponentially amplifying a specific target nucleic acid sequence. Themethod according to this aspect of the invention comprises contactingthe target nucleic acid sequence with a first forward primer nucleicacid, a second forward primer nucleic acid, a first reverse primernucleic acid and a second reverse primer nucleic acid under conditionswherein the primer nucleic acids specifically anneal with the targetnucleic acid sequence. One forward primer nucleic acid has a firstbond-forming reactive moiety and the other forward primer nucleic acidhas a second bond-forming reactive moiety. One reverse primer nucleicacid has a first bond-forming reactive moiety and the other reverseprimer nucleic acid has a second bond-forming reactive moiety. In someembodiments, the first or second bond-forming reactive moiety arethermally stable, for example at temperatures capable of denaturingdouble-stranded nucleic acids. The first forward primer nucleic acid andthe second forward primer nucleic acid are annealed to the targetnucleic acid sequence such that the reactive moiety of the first forwardprimer nucleic acid and the reactive moiety of the second forward primernucleic acid are juxtaposed. The first reverse primer nucleic acid andthe second reverse primer nucleic acid are annealed to the targetnucleic acid sequence such that the reactive moiety of the first reverseprimer nucleic acid and the reactive moiety of the second reverse primernucleic acid are juxtaposed. The reactive moiety of the first forwardprimer nucleic acid forms a covalent bond with the reactive moiety ofthe second forward primer nucleic acid to form a first ligation product,and the reactive moiety of the first reverse primer nucleic acid forms acovalent bond with the reactive moiety of the second reverse primernucleic acid to form a second ligation product. Thus, the first ligationproduct forms a duplex with the target nucleic acid sequence and thesecond ligation product forms a duplex with the target nucleic acidsequence. The duplexes are then disrupted, for example by thermaldenaturation, to form additional nucleic acid sequences which can serveas target nucleic acid sequences in the next cycle and the steps arerepeated to thereby amplify the target nucleic acid sequences.

Template nucleic acid sequences can be single-stranded ordouble-stranded. If the template nucleic acid is single-stranded, in thefirst cycle of the amplification reaction only one of the pairs ofprimers (either the forward primers or reverse primers) will be able tobind to the template nucleic acid and form a duplexed ligation product.Upon disruption of the ligation product, the complement of the templatenucleic acid will be present in the reaction mixture, thus allowing theother pair of primers to bind, such that all four primers are bound whenthe next cycle is initiated.

In some embodiments, the first bond-forming reactive moiety is anucleophilic moiety and the second bond-forming reactive moiety is anelectrophilic moiety. In some embodiments, the first bond-formingreactive moiety is an electrophilic moiety and the second bond-formingreactive moiety is a nucleophilic moiety. In some embodiments, the thirdbond-forming reactive moiety is a nucleophilic moiety and the fourthbond-forming reactive moiety is an electrophilic moiety. In someembodiments, the third bond-forming reactive moiety is an electrophilicmoiety and the fourth bond-forming reactive moiety is a nucleophilicmoiety. The nucleophilic or electrophilic moieties may be, for example,thermally stable.

In some embodiments, one forward or reverse primer nucleic acidcomprises a dye or detectable group. In some embodiments, one forward orreverse primer nucleic acid comprises a FRET donor fluorophore and/orthe other forward or reverse primer nucleic acid comprises a FRETacceptor fluorophore, and the ligation products are detected by FRET. Insome embodiments, the dye or detectable group is quenched by a quenchingmoiety in which annealing and autoligation separates the quenchingmoiety from the dye or detectable group before the ligated product isdetected.

In some embodiments the forward and reverse primer nucleic acids containneither a dye nor a detectable group, and the ligation products aredetected by double-stranded nucleic acid (e.g. dsDNA) binding dyes.

In a second aspect, the invention provides reagent compositions foramplifying a target nucleic acid sequence. In some embodiments, theinvention provides reagent compositions for linearly amplifying aspecific target nucleic acid sequence. In some embodiments, theinvention provides reagent compositions for exponentially amplifying aspecific target nucleic acid sequence. In some embodiments, a reagentcomposition according to the invention comprises a first forward primernucleic acid having a first bond-forming reactive moiety. In someembodiments, a reagent composition according to the invention comprisesa second forward primer nucleic acid having a second bond-formingreactive moiety. In some embodiments, a reagent composition according tothe invention comprises a first reverse primer nucleic acid having athird bond-forming reactive moiety. In some embodiments, a reagentcomposition according to the invention comprises a second reverse primernucleic acid having a fourth bond-forming reactive moiety. For example,the reactive moieties are thermally stable.

In some embodiments, the first bond-forming reactive moiety forms achemical bond with the second bond-forming reactive moiety, when thefirst forward primer nucleic acid and the second forward primer nucleicacid are juxtaposed by annealing with a target nucleic acid and when thefirst reverse primer and the second reverse primer nucleic acid arejuxtaposed by annealing with a target nucleic acid. In some embodiments,the first bond-forming reactive moiety is an alkyne (for example ahexynyl or octadiynyl group) and the second bond-forming reactive moietyis an azide. In some embodiments, the first bond-forming reactive moietyis an azide and the second bond-forming reactive moiety is an alkyne(for example a hexynyl or octadiynyl group). In some embodiments, oneforward or reverse primer nucleic acid comprises a dye or detectablegroup. In some embodiments, one forward or reverse primer nucleic acidcomprises a FRET donor fluorophore and/or the other forward or reverseprimer nucleic acid comprises a FRET acceptor fluorophore. In someembodiments, the dye or detectable group is quenched by a quenchingmoiety in which annealing and autoligation separates the quenchingmoiety from the dye or detectable group before the ligated product isdetected.

In some embodiments the forward and reverse primer nucleic acids containneither a dye nor a detectable group, and the ligation products aredetected by double-stranded nucleic acid binding dyes. For example, thereagent composition comprises a double-stranded nucleic acid (e.g.dsDNA) binding dye.

In a third aspect, the invention provides a kit for amplifying a targetnucleic acid sequence. In some embodiments, the invention provides a kitfor linearly amplifying a specific target nucleic acid sequence. In someembodiments, the invention provides a kit for exponentially amplifying aspecific target nucleic acid sequence. The kit according to this aspectof the invention comprises a first forward primer nucleic acid, a secondforward primer nucleic acid, a first reverse primer nucleic acid, and asecond reverse primer nucleic acid. In the kit according to this aspectof the invention, the first forward primer nucleic acid, the secondforward primer nucleic acid, the first reverse primer nucleic acid, andthe second reverse primer nucleic acid are as described for the secondaspect according to the invention.

Non-limiting examples of reagents and methods according to the inventionare shown in FIGS. 1, 2, 4, and 5, which illustrate the strategy andexpected results from two rounds of ACR, in which four double-strandedproducts are generated from the amplification of a single targetsequence. Forward ACR Primer 1 and Reverse ACR Primer 1 both contain abond-forming reactive moiety at the 3′ end. Forward ACR Primer 2 andReverse ACR Primer 2 both contain a bond-forming reactive moiety at the5′ end. When forward and reverse primers are annealed in tandem totemplate, the juxtaposition of the bond-forming reactive moietiesresults in a DNA-templated autoligation reaction without any enzymes ornucleotides. Primers annealed in tandem have higher melting temperaturedue to stabilizing base-pair stacking interactions between thetandemly-aligned oligos. See Lane, M. J., Paner, T., Kashin, I.,Faldasz, B. D., Li, B., Gallo, F. J. & Benight, A. S. (1997). Thethermodynamic advantage of DNA oligonucleotide ‘stacking hybridization’reactions: energetics of a DNA nick. Nucleic Acids Research, 25(3),611-617. ACR can be performed at annealing temperatures that favor theformation of primer/template heteroduplexes over primer dimers inhomoduplexes. The resulting autoligation products are used as templatesin subsequent rounds of amplification.

Non-limiting examples of bond-forming reactive moieties include moietieswhich participate in cycloaddition reactions, including azides andalkynes which participate in ‘click’ cycloaddition reactions. Otherexamples of possible bond-forming reactive moieties include thiolnucleophilic and bromoacetate electrophilic moieties, which are commongeneric chemistries that are commercially available. The preparation,protocol, and application of the 3′-thionucleoside thiol as athermal-stable nucleophile are well documented in the literature. See,for example, Ghalia Sabbagh, Kevin J. Fettes, Rajendra Gosain, Ian A.O'Neil and Richard Cosstick (2004). Synthesis of phosphorothioamiditesderived from 3′-thio-3′-deoxythymidine and 3′-thio-2′,3′-dideoxycytidineand the automated synthesis of oligodeoxynucleotides containing a3′-S-phosphorothiolate linkage. Nucleic Acids Research, 32(2) 495-501;Meena, Mui Sam, Kathryn Pierce, Jack W. Szostak, and Larry W.McLaughlin. (2′,3′-Dideoxy-3′-Thionucleoside Triphosphates: Synthesesand Polymerase Substrate Activities. Supporting Information; Miller, G.P., Silverman, A. P. & Kool, E. (2008). New, stronger nucleophiles fornucleic acid-templated chemistry: Synthesis and application influorescence detection of cellular RNA. Bioorganic & medicinalchemistry, 16(1), 56-64; Meena, Mui Sam, Kathryn Pierce, Jack W.Szostak, and Larry W. McLaughlin. (2007).(2′,3′-Dideoxy-3′-Thionucleoside Triphosphates: Syntheses and PolymeraseSubstrate Activities. Organic Letters. 9(6): 1161-1163; and Sengen Sun,Aiichiro Yoshids, and Joseph A. Piccirilli. (1997). Synthesis of3′-thioribonucleosides and their incorporation into oligoribonucleotidesvia phosphoramidite chemistry. RNA. 3: 1352-1363.

FIGS. 12-14 illustrate other non-limiting examples of bond-formingreactive moieties that include hexynyl and octadiynyl alkynes toconjugate with an azide bond-forming reactive moiety using cycloadditionchemistries to generate a covalent carbon-heteroatom bond betweenspecies to form a triazol conjugate.

FIG. 15 illustrates non-limiting examples ACR primer sequencescontaining an azide bond-forming reactive moiety (3AzideN), and hexynyl(5Hexynyl) and octadiynyl (55OCTdU) alkyne bond-forming reactivemoieties using a fluorescein (iFluorT) and TAMRA (i6-TAMN) detectiongroups in FRET for detection. This illustrates that a variety ofchemistries with auto-reactive moieties are available for ACR.

Non-limiting examples of bond-forming nucleophilic moieties includeazides, cyclooctyne, phosphorodithioate, phosphorotrithioate,2′,3′-cyclic phosphate, amino-deoxyribonucleosides, thiol, amino,hydrazine, and hydrazide. Non-limiting examples of bond-formingelectrophilic moieties include alkynes, tetrazine, bromide, iodide,chloride, maleimide, dabsylate, disulfides, tosylate, isothiocyanate,NHS ester, imidoester, PFP ester, alkyl azide, aryl azide, isocyanate,nitrophenyl mono- or di-ester, aldehyde, and epoxy.

Forward ACR Primer 1 and Reverse ACR Primer 2 and Forward ACR Primer 2and Reverse ACR Primer 1 are complementary pairs, which increase thespecificity of the reaction by sequestering the primers in duplexesuntil dsDNA templates outcompete the formation of primer homoduplexes byannealing to the primers. Because tandemly-annealed oligos on a templatehave significantly higher melting temperatures than individual oligosannealed to the same template, due to stabilizing base-pair stackinginteractions between the tandemly-aligned oligos, ACR can be performedat annealing temperatures that favor the formation of primer/templateheteroduplexes over homoduplexed primer sets.

For purposes of the invention, a “primer nucleic acid” is anoligonucleotide used in the method according to the invention to form alonger oligonucleotide via autoligation to another primer nucleic acid.Primer nucleic acids may be from about 5 to about 35 nucleotides inlength, for example from about 5 to about 25, about 5 to about 20, about5 to about 18, or about 10 to about 18 nucleotides. The autoligationreaction occurs when the primer nucleic acids are annealed to a targetnucleic acid sequence such that a first bond-forming reactive moiety ofone primer nucleic acid is juxtaposed with a second bond-formingreactive moiety of another primer nucleic acid. In some embodiments thefirst bond-forming reactive moiety is at a terminus (5′ or 3′) of oneprimer nucleic acid and the second bond-forming reactive moiety is at anopposite terminus of the other primer nucleic acid. The terms “firstbond-forming reactive moiety” and “second bond-forming reactive moiety”refer to chemical functional groups that are capable of reacting witheach other to form a covalent bond.

Non-limiting examples of first bond-forming reactive moieties includeazides, cyclooctyne, phosphorodithioate, phosphorotrithioate,2′,3′-cyclic phosphate, amino-deoxyribonucleosides, thiol, amino,hydrazine, and hydrazide. In some embodiments, the first bond-formingreaction is an azide. In certain embodiments, the first bond-formingreactive moiety is a nucleophile. In some embodiments, the 3′ terminalnucleophile is a 3′-thionucleoside.

Non-limiting examples of second bond-forming reactive moieties includealkynes, tetrazine, bromide, iodide, chloride, maleimide, dabsylate,disulfides, tosylate, isothiocyanate, NHS ester, imidoester, PFP ester,alkyl azide, aryl azide, isocyanate, nitrophenyl mono- or di-ester,aldehyde, and epoxy. In some embodiments, the second bond-formingreactive moiety is an alkyne, for example an octadiynyl or hexynylgroup. In certain embodiments, the second bond-forming reactive moietyis an electrophile. In some embodiments, the 5′-terminal electrophile isa 5′-bromoacetylnucleoside.

Amplification of a double-stranded target nucleic acid sequence requiresdisruption of duplex target sequence. In some embodiments, disruptionoccurs by thermally denaturing double-stranded target nucleic acidsequence by raising the temperature above the melting temperature.

Reaction efficiency is enhanced when the bond-forming moieties used arethermally stable. In this context, the term “thermally stable” meansthat the reactivity of a bond-forming moiety is not destroyed orfunctionally compromised to such an extent that the desired reaction nolonger occurs with sufficient efficiency at temperatures required todenature the target sequence.

In some embodiments, a dye or detectable group is used to detect theligated products formed by annealing and autoligation. Non-limiting dyesand detectable groups include, without limitation, the groups shown inTable I below.

TABLE I Detectable Dyes and Groups (E)-Stilbene (Z)-Stilbene1-Chloro-9,10-bis(phenylethynyl)anthracene2-Chloro-9,10-bis(phenylethynyl)anthracene2-Chloro-9,10-diphenylanthracene 5,12-Bis(phenylethynyl)naphthacene7-Aminoactinomycin D 7-Aminoactinomycin D (7-AAD)7-Hydroxy-4-methylcoumarin 8-Anilinonaphthalene-1-sulfonate9,10-Bis(phenylethynyl)anthracene Acridine orange Acridine yellow AlexaFluor Alexa Fluor 350 dye, 7-amino-4-methylcoumarin (AMC) Alexa Fluor405 dye Alexa Fluor 430 dye Alexa Fluor 488 dye Alexa Fluor 514 dyeAlexa Fluor 532 dye Alexa Fluor 546 dye Alexa Fluor 555 dye Alexa Fluor568 dye Alexa Fluor 594 dye Alexa Fluor 610 dye Alexa Fluor 633 dyeAlexa Fluor 635 dye Alexa Fluor 647 dye Alexa Fluor 660 dye Alexa Fluor680 dye Alexa Fluor 700 dye Alexa Fluor 750 dye Alexa Fluor 790 dyeAllophycocyanin ATTO dyes Auramine-rhodamine stain BCECF indicatorBenzanthrone BHQ-1 BHQ-2 BHQ-3 Bimane Blacklight paint blue fluorescentproteins BOBO-1, BO-PRO-1 BODIPY 630/650 dye BODIPY 650/665 dye BODIPYdye BODIPY FL dye BODIPY TMR-X dye BODIPY TR-X dye Brainbow CalceinCalcium Crimson indicator Calcium Green indicators Calcium Orangeindicator Carboxy SNARF indicators Carboxyfluorescein Carboxyfluoresceindiacetate succinimidyl ester Carboxyfluorescein succinimidyl esterCascade Blue dye Cascade Yellow dye Chemiluminescent ColorimetricCoumarin Cy-3 Cy-5 Dabcyl DAPI Dark quencher DDQ-I DDQ-II Di-8-ANEPPS,Di-4-ANEPPS DiA DiD (DiIC18(5)) DiI (DiIC18(3)) DiO (DiOC18(3)) DiOC6DiR (DiIC18(7)) DyLight Fluor Eclipse ELF 97 alcohol Eosin ER TrackerBlue-White DPX EthD-1 Ethidium bromide excimer/exciplex partner exciplexdyes FAM Fluo-3 indicator Fluo-4 Fluo-4 indicator FluoProbes FluoresceinFluorescein isothiocyanate Fluorescein, FITC Fluoro-Jade stainfluorophore-quencher couples, FM 1-43, FM 1-43FX FM 4-64, FM 4-64FX FuraRed indicator Fura-2 indicator Fura-2-acetoxymethyl ester gold nanoparticles Green fluorescent protein HEX Hoechst 33258, Hoechst 33342Indian yellow Indo-1 inorganic quantum dots Iowa Black FQ Iowa Black RQJC-1 JC-9 JOE LC red 640 LC red 705 Lissamine rhodamine B Lucifer yellowLucifer yellow CH Luciferin LysoSensor Blue DND-167 LysoSensor GreenDND-153, DND-189 LysoSensor Yellow/Blue DND-160 (PDMPO) LysoTrackerGreen LysoTracker Red Magnesium Green indicator Marina Blue dyeMerocyanine MGB groups MitoTracker Green FM MitoTracker Orange CMTMRosMitoTracker Red CMXRos Monobromobimane NBD amines NED NeuroTrace 500/525green-fluorescent Nissl stain Nile blue Nile red Optical brightenerOregon Green 488 dye and Oregon Green 488 BAPTA Oregon Green 514 dyePacific Blue dye Pacific Orange dye Perylene Phloxine PhycobilinPhycoerythrin Phycoerythrobilin POPO-1, PO-PRO-1 Propidium iodidePyranine QSY-21 QSY-7 R-phycoerythrin red fluorescent proteins ResorufinRH 414 Rhod-2 indicator Rhodamine Rhodamine 110 Rhodamine 123 Rhodamine123 Rhodamine 6G Rhodamine Green dye Rhodamine Red dye RiboGreen RoGFPROX Rubrene SERRS-active fluorescence dyes Sodium Green indicatorSulforhodamine 101 Sulforhodamine B SYBR Green Synapto-pHluorin SYTOblue-fluorescent nucleic acid stains 40, 41, SYTO blue-fluorescentnucleic acid stains 44, 45 SYTO green-fluorescent nucleic acid stains11, 14, 15, 20, SYTO green-fluorescent nucleic acid stains 12, 13, 16,21, SYTO orange-fluorescent nucleic acid stains 80, 81, 82, SYTOorange-fluorescent nucleic acid stains 84, SYTO red-fluorescent nucleicacid stains 17, 59, SYTO red-fluorescent nucleic acid stains 60, 62,SYTOX Blue nucleic acid stain SYTOX Green nucleic acid stain SYTOXOrange nucleic acid stain TAMRA TET Tetramethylrhodamine, Rhodamine BTetraphenyl butadiene Tetrasodium tris(bathophenanthroline Texas RedTexas Red-X dye Titan yellow TMR TOTO-1, TO-PRO-1 TOTO-3, TO-PRO-3 TSQUmbelliferone X-rhod-1 indicator Yellow fluorescent protein YOYO-1,YO-PRO-1 YOYO-3, YO-PRO-3

In some embodiments, the first forward primer and second forward primeror the first reverse primer are conjugated to dyes that are,respectively, a donor dye and an acceptor dye for FRET. Alternatively,the first forward primer and second forward primer or the first reverseprimer are conjugated to dyes that are, respectively, an acceptor dyeand a donor dye for FRET. Alternatively, the donor and acceptor dyes forFRET may be, respectively, on the second reverse primer and the firstreverse primer or the second forward primer. Alternatively, the secondreverse primer and the first reverse primer or the second forward primerare conjugated to dyes that are, respectively, an acceptor dye and adonor dye for FRET. Alternatively, the first forward primer and secondforward primer are conjugated to dyes that are, respectively, anacceptor dye and a donor dye, and the second reverse primer and thefirst reverse primer are conjugated to dyes that are, respectively, anacceptor dye and a donor dye for FRET. Alternatively, the first forwardprimer and second forward primer are conjugated to dyes that are,respectively, a donor dye and an acceptor dye, and the second reverseprimer and the first reverse primer are conjugated to dyes that are,respectively, a donor dye and an acceptor dye for FRET. In someembodiments, the donor and acceptor dyes are spaced from about 1 toabout 20 nucleotides apart within the autoligation product, for examplewithin about 1 to about 15, about 1 to about 10, about 1 to about 5, orabout 1 to about 3 nucleotides. In some embodiments, the donor dye isFAM and the acceptor dye is Texas Red.

In some embodiments, the dye or detectable group is quenched by aquenching moiety in which annealing and autoligation separates thequenching moiety from the dye or detectable group before the ligatedproduct is detected.

In some embodiments the forward and reverse primer nucleic acids containneither a dye nor a detectable group, and the ligation products aredetected by double-stranded nucleic acid binding dyes.

In some embodiments, a method of the invention is used to detect thepresence or absence of a mutation, for example a SNP mutation, in abiological sample. Generally, the test sample can be biological and/orenvironmental samples. Biological samples may be derived from human,other animals, or plants, body fluid, solid tissue samples, tissuecultures or cells derived therefrom and the progeny thereof, sections orsmears prepared from any of these sources, or any other samplessuspected to contain the target nucleic acids. Biological samplesinclude body fluids including but not limited to blood, urine, spinalfluid, cerebrospinal fluid, sinovial fluid, amniotic fluid, semen, andsaliva. Other types of biological sample may include food products andingredients such as vegetables, dairy items, meat, meat by-products, andwaste. Biological samples also include plant tissue such as seed or leaftissue. Environmental samples are derived from environmental materialincluding but not limited to soil, water, sewage, cosmetic, agriculturaland industrial samples.

In some embodiments, a method of the invention is used to perform highresolution melt curve analysis (HRM). DNA melt curve analysis can revealthe number of DNA species or purity of an amplification reaction, andthus is often used as a more convenient alternative to gelelectrophoresis to confirm the specificity of ACR. According to oneembodiment, the nucleic acid detection is associated with highresolution melt curve analysis (HRM). Compared to regular DNA melt curveanalysis, HRM can yield more information on the amplified DNA product,including the capability for point mutation detection (SNP), zygositytesting and epigenetics analysis. Like regular DNA melt curve analysis,HRM is a post-ACR product analysis method. In HRM, a target nucleic acidis first amplified by ACR in the presence of a DNA binding dye and thenthe PCR product-dye complex is slowly melted as the fluorescence changeis monitored to generate a standard DNA melt curve. The procedure isrepeated with additional target nucleic acid(s) to generate additionalmelt curve(s). The additional melt curve(s) are compared with thestandard curve to yield minor differences that may be indicative ofmutation site(s) in the target nucleic acid sequences (U.S. Pat. Nos.7,387,887; 7,456,281; and 7,582,429).

The invention provides for systems that can be used to detect targetanalytes, such as nucleic acids. The system can include at least onedetector (e.g., a spectrometer, etc.) that detects a signal that isindicative of a target analyte. For example, the system can include adetector for measuring an optical signal, such as fluorescence. Inaddition, the system can include at least one thermal modulator (e.g., athermal cycling device, etc.) operably connected to a container or solidsupport to modulate temperature of a sample. The thermal modulator canbe used for performing nucleic acid amplification methods, melting curveanalysis, and/or hybridization assays.

Detectors can be structured to detect detectable signals produced, e.g.,in or proximal to another component of the given assay system (e.g., incontainer, on a solid support, etc.). Suitable signal detectors that areoptionally utilized, or adapted for use, herein detect, e.g.,fluorescence, phosphorescence, radioactivity, absorbance, refractiveindex, luminescence, mass, or the like. Detectors optionally monitor oneor a plurality of signals from upstream and/or downstream of theperformance of, e.g., a given assay step. For example, detectorsoptionally monitor a plurality of optical signals, which correspond toreal-time events. Example detectors or sensors include photomultipliertubes, CCD arrays, optical sensors, temperature sensors, pressuresensors, pH sensors, conductivity sensors, scanning detectors, or thelike. More specific exemplary detectors that are optionally utilized inperforming the methods of the invention include, e.g., resonance lightscattering detectors, emission spectroscopes, fluorescencespectroscopes, phosphorescence spectroscopes, luminescencespectroscopes, spectrophotometers, photometers, and the like. Detectorsare also described in, e.g., Skoog et al., Principles of InstrumentalAnalysis, 5^(th) Ed., Harcourt Brace College Publishers (1998) andCurrell, Analytical Instrumentation: Performance Characteristics andQuality, John Wiley & Sons, Inc. (2000), both of which are incorporatedby reference.

The systems of the invention can include controllers that are operablyconnected to one or more components (e.g., detectors, thermalmodulators, fluid transfer components, etc.) of the system to controloperation of the components. More specifically, controllers can beincluded either as separate or integral system components that areutilized, e.g., to receive data from detectors, to effect and/orregulate temperature in the containers, to effect and/or regulate fluidflow to or from selected containers, or the like. Controllers and/orother system components is/are optionally coupled to an appropriatelyprogrammed processor, computer, digital device, or other informationappliance (e.g., including an analog to digital or digital to analogconverter as needed), which can function to instruct the operation ofthese instruments in accordance with preprogrammed or user inputinstructions, receive data and information from these instruments, andinterpret, manipulate and report this information to the user.Controllers are available from various commercial sources.

Any controller or computer optionally includes a monitor, which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display, etc.), or others.Computer circuitry is often placed in a box, which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser.

The computer can include appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of one or more controllers to carry out thedesired operation. The computer then receives the data from, e.g.,sensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as controlling fluid flow regulators in responseto fluid weight data received from weight scales or the like.

In some embodiments, the invention provides integrated systems forperforming ACR and for making T_(m) determinations. The systems caninclude instrumentation and tools for interpreting and analyzingcollected data, especially including tools for determining quantity ofamplified nucleic acids and for deriving T_(m). These tools can includealgorithms and/or the ability to electronically store information (e.g.,collected fluorescence data, predetermined T_(m) correlations, etc).Each part of an integrated system can be functionally interconnected,and in some cases, physically connected. In some embodiments, theintegrated system is automated, where there is no requirement for anymanipulation of the sample or instrumentation by an operator followinginitiation of the ACR or T_(m) analysis.

A system of the invention can include instrumentation. For example, theinvention can include a detector such as a fluorescence detector (e.g.,a fluorescence spectrophotometer). A detector or detectors can be usedin conjunction with the invention, e.g., to monitor/measure the emissionfrom a light emitting moiety, such as a nucleic acid dye. A detector canbe in the form of a multiwell plate reader to facilitate thehigh-throughput capacity of the assays described herein.

In some embodiments, the integrated system includes a thermal cyclingdevice, or thermocycler, for the purpose of controlling the temperatureof the T_(m) melting analysis or for modulating the temperature forperforming nucleic acid ampliflication. In some embodiments, the thermalcycling device and the detector are an integrated instrument, where thethermal cycling and emission detection (e.g., fluorescence detection)are performed in the same device.

A detector, e.g., a fluorescence spectrophotometer, can be connected toa computer for controlling the spectrophotometer operational parameters(e.g., wavelength of the excitation and/or wavelength of the detectedemission) and/or for storage of data collected from the detector (e.g.,fluorescence measurements during a melting curve analysis). The computermay also be operably connected to the thermal cycling device to controlthe temperature, timing, and/or rate of temperature change in thesystem. The integrated computer can also contain the “correlationmodule” where the data collected from the detector is analyzed and wherethe T_(m) of the target hybridization complex and/or the concentrationof amplified or target nucleic acid is determined. In some embodiments,the correlation module comprises a computer program that calculates theT_(m) or the concentration of nucleic acid based on the fluorescencereadings from the detector, and in some cases, optionally derivessequence and/or genotype information of an unknown sample based on theT_(m) and/or ACR result. In some embodiments, the correlation modulecompares the T_(m) of the unknown sample with a database (or table) ofT_(m) values for known sequences and/or genotypes to make a correlationbetween the T_(m) of the unknown sample and the sequence or genotype ofthe unknown sample.

In some aspects, a system of the invention for the determination of aT_(m) of a hybridization complex and/or for performing ACR comprises areagent composition, a thermal control device for regulating thetemperature reaction over a range of temperatures, and a detector formeasuring the signal from the melting reaction over the range oftemperatures. In some cases, the system also includes a correlationmodule that is operably coupled to the detector and receives signalmeasurements, where the correlation module correlates the signalintensity with the concentration of the target analyte or the meltingtemperature of the target analyte.

The following examples are intended to further illustrate certainembodiments of the invention and are not to be construed to limit thescope of the invention.

Example 1 ACR Amplification Method

The method and expected results from two rounds of ACR, in which fourdouble-stranded products are generated from the amplification of asingle nucleic acid target sequence are shown (FIG. 1). Forward ACRPrimer 1 and Reverse ACR Primer 1 are both labeled with a firstbond-forming reactive moiety at the 3′ end (BFRM 1). Forward ACR Primer2 and Reverse ACR Primer 2 are both labeled with a second bond-formingreactive moiety at the 5′ end (BFRM 2). When forward and reverse primersare annealed in tandem to template, the juxtaposition of the first andsecond bond-forming reactive moieties results in a nucleicacid-templated autoligation reaction without any enzymes or nucleotides.Primers annealed in tandem have higher melting temperature due tostabilizing base-pair stacking interactions between the tandemly-alignedoligos. See Lane, M. J., Paner, T., Kashin, I., Faldasz, B. D., Li, B.,Gallo, F. J. & Benight, A. S. (1997). The thermodynamic advantage of DNAoligonucleotide ‘stacking hybridization’ reactions: energetics of a DNAnick. Nucleic Acids Research, 25(3), 611-617. ACR can be performed atannealing temperatures that favor the formation of primer/templateheteroduplexes over primer dimers in homoduplexes. The resultingautoligation products are used as templates in subsequent rounds ofamplification to generate exponential growth of double-strandedproducts. A variety of bond-forming reactive moieties can be used withthe methods described herein, which are not limited to any specificbond-forming chemistry.

Example 2 ACR Detection with Dyes

An example using detection dyes and expected results from two rounds ofACR, in which four double-stranded products are generated from theamplification of a single nucleic acid target sequence are shown, anddetection with double-stranded nucleic acid binding dyes (DBD) (FIG. 2).The resulting autoligation products are detected by DBDs whendouble-stranded. In this example, detection by DBD binding todouble-stranded nucleic acids is achieved at temperatures below (<) themelting temperature (T_(m)) of the ACR products and above (>) the T_(m)of the self-ligating ACR primers. A variety of bond-forming reactivemoieties can be used with the methods described herein, which are notlimited to any specific bond-forming chemistry.

Example 3 ACR Detection with SYBR Green I in Real-Time

The results of ACR self-ligation reactions using a dye with dsDNAnucleic acid template using duplexed tandem primers or single tandemprimers and SYBR Green I for detection of binding to the duplexes formedfrom amplification reactions in the presence or absence of a SNPmutation (FIG. 3). Each trace represents an average of 4 replicatesmeasured across 26 cycles. Each reaction contained a 1000-fold molarexcess of self-ligating primers over a dsDNA primer template. SYBR GreenI binding to duplexes formed between the autoligation products andcomplementary strands of template DNA was used to monitor the increasein double-stranded amplification products from a single pair of ACRprimers (“Single Tandem Primers”), or a pair of ACR primers with theircomplementary primers (“Duplexed Tandem Primers”). Template carrying aSNP mutation (“SNP Mutation”) was used to determine the specificity ofthe reaction. Omitted template (“No Template Control”) was used as anegative control for the reaction.

Reactions were performed using the strategy shown in FIG. 2, using7-mers (Forward ACR Primer 1 and Reverse ACR Primer 2) and 13-mers(Forward ACR Primer 2 and Reverse ACR Primer 1). Forward ACR Primer 1and Reverse ACR Primer 1 contain a phosphoromono-thioate esternucleophile (BFRM 1) at the 3′-terminus, while Forward ACR Primer 2 andReverse ACR Primer 2 contain a dabsylate electrophile (BFRM 2) at the 5′end. SYBR Green I was used to monitor DNA amplification ofdouble-stranded ACR products. In each reaction, the concentration of thenucleophilic primer was 200 nM, and the electrophilic primer was 100 nM.A 28-mer dsDNA nucleic acid template was added at 0.01 nM concentration.Reactions were thermocycled on an ABI PRISM® 7900HT Sequence DetectionSystem, using a touchdown annealing strategy from 22° C. to 10° C., anda 95° C. melting temperature. The clipped, normalized baselined data(delta Rn, for cycles 3-8) was exported into Excel, and the plots weresmoothed by a 6-point rolling average of the data. FIG. 2 illustratesspecificity of the methods disclosed herein for detection of SNPs.

Example 4 ACR Detection with Unquenched Detection Group

An example using a fluorophore/quenched detection group and expectedresults from two rounds of ACR, in which four double-stranded productsare generated from the amplification of a single nucleic acid targetsequence, and detection with a detection group (Fluor) in which aquenching moiety (Quen) is separated from the detection group afterannealing and autoligation separates the quenching moiety from thedetectable group before the ligated product is detected (FIG. 4).Forward 1 and Reverse 1 ACR primers both contain BFRM 1 at the 3′ end.Forward 2 and Reverse 2 ACR primers both contain a quenching BFRM 2 atthe 5′ end, and an internal signal fluorophore (Fluor), which isquenched by BFRM 2. When an excess of ACR primers are annealed in tandemto their limiting template strands, the juxtaposition of the BFRM 1 andBFRM 2 groups causes the displacement of BFRM 2 by BFRM 1 resulting innucleic acid-templated autoligation of the tandem ACR primers on eachstrand.

The resulting Forward 1/2 and Reverse 1/2 ligation products are used astemplates in subsequent rounds of amplification, in which the signalincreases due to more Fluor becoming un-quenched at each cycle. ACRForward 1 and ACR Reverse 2 primers, and ACR Forward 2 and ACR Reverse 1primers are complementary pairs, which increase the specificity of thereaction by sequestering the primers in duplexes until nucleic acidtemplates outcompete the formation of ACR primer homoduplexes byannealing to the primers. Because tandemly-annealed primers on atemplate have significantly higher melting temperatures than individualprimers annealed to the same template, due to stabilizing base-pairstacking interactions between the tandemly-aligned primers, ACR can beperformed at annealing temperatures that favor the formation of ACRprimer/template heteroduplexes over homoduplexed primer sets. See Lane,M. J., Paner, T., Kashin, I., Faldasz, B. D., Li, B., Gallo, F. J. &Benight, A. S. (1997). The thermodynamic advantage of DNAoligonucleotide ‘stacking hybridization’ reactions: energetics of a DNAnick. Nucleic Acids Research, 25(3), 611-617. A variety of bond-formingreactive moieties can be used with the methods described herein, whichare not limited to any specific bond-forming chemistry.

Example 5 ACR Detection with FRET Detection Groups

An example of FRET detection and expected results from two rounds ofACR, in which four double-stranded products are generated from theamplification of a single nucleic acid target sequence, and detectionwith a first fluorophore detection group (F1) and a second fluorophoredetection group (F2) by FRET (FIG. 5). Forward ACR Primer 1 and ReverseACR Primer 1 both contain a BFRM 1 at the 3′ end. Forward ACR Primer 2and Reverse ACR Primer 2 both contain a BFRM 2 at the 5′ end. Whenforward and reverse primers are annealed in tandem to template, thejuxtaposition of the BFRM 1 and BFRM 2 groups results in a nucleicacid-templated autoligation reaction without any enzymes or nucleotides.Primers annealed in tandem have higher melting temperature due tostabilizing base-pair stacking interactions between the tandemly-alignedoligos. See Lane, M. J., Paner, T., Kashin, I., Faldasz, B. D., Li, B.,Gallo, F. J. & Benight, A. S. (1997). The thermodynamic advantage of DNAoligonucleotide ‘stacking hybridization’ reactions: energetics of a DNAnick. Nucleic Acids Research, 25(3), 611-617. ACR can be performed atannealing temperatures that favor the formation of primer/templateheteroduplexes over primer dimers in homoduplexes. The resultingautoligation products are used as templates in subsequent rounds ofexponential amplification. A variety of bond-forming reactive moietiescan be used with the methods described herein, which are not limited toany specific bond-forming chemistry.

Example 6 ACR Detection with Stained Polyacrylamide Gels

An example of results of ACR self-ligation reactions using unstained andstained polyacrylamide gels after an autoligation reaction for detectionwhere the first forward primer nucleic acid contains a firstbond-forming reactive moiety and a second forward primer nucleic acidcontains a second bond-forming reactive moiety in which the secondforward primer is labeled with FAM (FIG. 6). Reactions were performedusing unlabeled Forward ACR Primer 1 nucleophile (GCAACGACCGTTCCGT-SH)(SEQ ID NO: 1) and labeled Forward ACR Primer 2 electrophile(BrAc-TCAAT(FAM)ACTGCGCAGCC) (SEQ ID NO: 2). Increasing ssDNA nucleicacid template was added to reactions in a molar excess. FIG. 6 shows theefficiency of the forward ACR primers for autoligation by titrating inincreasing amounts of single-stranded complementary nucleic acidtemplate. Lane 1 of each panel is the no-template control. The threepanels show the same gel using different detection systems. The leftpanel shows FAM fluorescence using the Typhoon Trio+ imaging system. Themiddle panel shows SYBR fluorescence using the Typhoon Trio+ imagingsystem after staining the gel with SYBR Green I. The right panel showsSYBR fluorescence using the Alphalmager imaging system after stainingthe gel with SYBR Green I. Different detection media and differentdetection platforms can be used with the methods herein, which are notlimited to a single detection medium or detection platform.

Example 7 Unlabeled ACR Primer Detection with Stained PolyacrylamideGels

An example of results of ACR self-ligation reactions using a stainedpolyacrylamide gel after an autoligation reaction with a first reverseprimer nucleic acid containing a first bond-forming reactive moiety anda second reverse primer nucleic acid containing a second bond-formingreactive moiety in which the first and second reverse primers areunlabeled. Reactions were performed using unlabeled Reverse ACR Primer 1nucleophile (GGCTGCGCAGTAT-SH) (SEQ ID NO: 3) and unlabeled Reverse ACRPrimer 2 electrophile (BrAc-TGAACGGAACGGTCGTTGC) (SEQ ID NO: 4).Increasing ssDNA nucleic acid template was added to reactions in a molarexcess (lanes 2-5). Lane 1 of each panel is the no-template control.FIG. 7 shows 2 panels of the same gel using different detection systems.The left panel shows SYBR fluorescence using the Typhoon Trio+ imagingsystem after staining the gel with SYBR Green I. The right panel showsSYBR fluorescence using the Alphalmager imaging system after stainingthe gel with SYBR Green I.

Example 8 ACR Primer FRET Detection with Unstained Polyacrylamide Gels

An example of results of ACR showing FAM/Texas Red FRET fluorescence ofACR reactions on an unstained (FIG. 8). Reactions were performed usingTexas Red-labeled Forward ACR Primer 1, FAM-labeled Forward ACR Primer2, and unlabeled reverse primers, with ssDNA nucleic acid as template.FIG. 8 shows FAM/Texas Red FRET fluorescence of reactions on anunstained 20% acrylamide+urea denaturing gel using the Typhoon Trio+imaging system. Lane 1 contains ssDNA template, and Lane 2 is theno-template control. The autoligation product was excited at 488 nM andthe fluorescence emission was detected at both 520 nM (FAM channel) and610 nM (Texas Red FRET channel) on the Typhoon Trio+ imaging system,demonstrating FRET detection after thermocycling.

Example 9 ACR Activity Enrichment

An example of results showing enrichment of ACR activity using differentfractions of electrophilic ACR primers (FIG. 9). Detection usedFAM/Texas Red FRET fluorescence of ACR reactions on an unstained 20%acrylamide+urea denaturing gel using the Typhoon Trio+ imaging system.FIG. 9 shows FAM/Texas Red FRET fluorescence of reactions on anunstained 20% acrylamide+urea denaturing gel using the Typhoon Trio+detection system. Reactions were performed using Texas Red-labeledForward ACR Primer 1 and FAM-labeled Forward ACR Primer 2 with ssDNAnucleic acid as template. Lanes 1 and 3 are the no-template controllanes. Lanes 2 and 4 contain ssDNA template. The autoligation reactionswere excited at 488 nM and the fluorescence emission was detected atboth 520 nM (FAM channel) and 610 nM (Texas Red FRET channel) on theTyphoon Trio+ detection system, demonstrating FRET detection.

Example 10 ACR FRET Enhancement

An example of results showing enhancement of ACR FRET using the samereactions with enriched ACR activity from FIG. 9 (Lanes 3 and 4) usingtwo different channels for detection: Non-FRET and FRET channels (FIG.10). Detection used FAM/Texas Red FRET fluorescence of ACR reactions onan unstained 20% acrylamide+urea denaturing gel using the Typhoon Trio+imaging system. Reactions were performed using Texas Red-labeled ForwardACR Primer 1 and FAM-labeled Forward ACR Primer 2 with ssDNA nucleicacid as template. Lanes 1 and 3 are the no-template control lanes. Lanes2 and 4 contain ssDNA template. The reactions were excited at 488 nM andthe fluorescence emission was detected at 520 nM (FAM channel) (Lanes 1and 2) and at 610 nM (Texas Red FRET channel) (Lanes 3 and 4) on theTyphoon Trio+ detection system, demonstrating FRET detection.

Examples 11A and 11B ACR Real-Time FRET Detection

An example of results showing real-time amplification plots on theLightCycler® 480 II using FAM/Texas Red FRET fluorescence in ACRreactions to demonstrate exponential amplification (FIG. 11A). Reactionswere performed using Texas Red-labeled Forward ACR Primer 1, FAM-labeledForward ACR Primer 2, and unlabeled reverse primers, with ssDNA nucleicacid as template. The normalized baselined data was exported into Excel,and the plots were smoothed by a 4-point rolling average of the data.The trace plot labeled “With Template” shows exponential amplificationof a reaction with template, DNA, and the “No Template” plot shows anegative no-template control. The same reactions were run on a SYBRGreen I stained 20% acrylamide+urea denaturing gel using the TyphoonTrio+ imaging system (FIG. 11 B). Lane 1 contains the no-templatecontrol, while Lane 2 shows the amplification product in the presence oftemplate. This illustrates that ACR can be detected in real-time, andtherefore ACR can be used for both end-point and quantitative analyses.

Example 12 ACR Bond-Forming Chemistry Through Cycloaddition

A non-limiting example of an ACR bond-forming chemistry throughcycloaddition with alkyne and azide moieties to generate a covalentcarbon-heteroatom bond between species to form a 1,2,3-triazoleconjugate (FIG. 12). The cycloaddition chemistry reaction couples andazide group with an alkyne group through a copper-catalyzed (Cu(I))reaction. The reaction is stable, irreversible, and has no sideproducts, and the bond-forming alkyne and azide moieties arecommercially available. See Rostovtsev, V. V., et al. (2002). A stepwisehuisgen cycloaddition process: copper(I)-catalyzed regioselective“ligation” of azides and terminal alkynes. Angew Chem Int Ed Engl.41(14): 2596-2599; Moses, J. E. and A. D. Moorhouse, (2007). The growingapplications of click chemistry. Chem Soc Rev. 36(8): 1249-1262.

Example 13 ACR Bond-Forming Reactive Moiety with Hexynyl

A non-limiting example of a bond-forming reactive moiety using a hexynylalkyne modification (FIG. 13). Hexynyl can be used to conjugate an ACRPrimer 2 to an ACR Primer 1 modified with an azide bond-forming reactivemoiety.

Example 14 ACR Bond-Forming Reactive Moiety with Octadiynyl

A non-limiting example of a bond-forming reactive moiety using a hexynylan octadiynyl alkyne modification (FIG. 14). Octadiynyl can be used toconjugate an ACR Primer 2 to an ACR Primer 1 modified with an azidebond-forming reactive moiety.

Example 15 ACR Primers Containing Azide and Alkyne Bond-Forming ReactiveMoieties

Non-limiting examples showing ACR primer sequences containing an azidebond-forming reactive moiety (3AzideN, Integrated DNA Technologies), andhexynyl (5Hexynyl, Integrated DNA Technologies) and octadiynyl (55OCTdU, Integrated DNA Technologies) alkyne bond-forming reactivemoieties to detect a mutation 18-599m in the maize GlutathioneS-Transferase (GST) gene (FIG. 15). See Rostovtsev, V. V., et al.(2002). A stepwise huisgen cycloaddition process: copper(I)-catalyzedregioselective “ligation” of azides and terminal alkynes. Angew Chem IntEd Engl. 41(14): 2596-2599; Moses, J. E. and A. D. Moorhouse, (2007).The growing applications of click chemistry. Chem Soc Rev. 36(8):1249-1262.

Detection is performed through FRET using a fluorescein (iFluorT) andTAMRA (i6-TAMN) detection groups.

Examples 16A and 16B ACR with Azide and Hexynyl Bond-Forming ReactiveMoieties

An example of results showing real-time amplification plots withcycloaddition on the LightCycler® 480 II using FAM/TAMRA FRETfluorescence in ACR reactions to demonstrate exponential amplification(FIG. 16A). Reactions were performed using FAM-labeled Forward ACRPrimer 1, TAMRA-labeled Forward ACR Primer 2, and unlabeled reverseprimers, with dsDNA nucleic acid as template. The non-baselined datawere exported into Excel. The trace plots show enhanced exponentialamplification profiles over thiol nucleophilic and bromoacetateelectrophilic moieties in ACR (FIGS. 11A and 11B), making ACR moreamenable to both end-point and quantitative analyses. The same reactionswere run on a 20% acrylamide+urea denaturing gel using the Typhoon Trio+imaging system (FIG. 16 B). Lanes 1-3 contain more concentrated catalystthan lanes 4-9 and show FRET-specific fluorescence of the ligated ACRprimers. This illustrates that azide and hexynyl bond-forming reactivemoieties have greater sensitivity with an enhanced exponentialamplification profile over thiol nucleophilic and bromoacetateelectrophilic moieties in ACR (FIGS. 11A and 11B), making ACR moreamenable to both end-point and quantitative analyses.

Example 17 ACR with Azide and Octadiynyl Bond-Forming Reactive Moieties

An example of results showing real-time amplification using azide andoctadiynyl bond-forming reactive moieties with FAM/TAMRA FRETfluorescence in ACR run on a 20% acrylamide+urea denaturing gel usingthe Typhoon Trio+ imaging system. FRET templated ligation is observeddown to 100 copies of template (lanes 2-7). This illustrates that azideand octadiynyl bond-forming reactive moieties have greater sensitivityover thiol nucleophilic and bromoacetate electrophilic moieties in ACR(FIGS. 11A and 11B), making ACR more amenable to both end-point andquantitative analyses.

Example 18 Thermostability of ACR Primers

Reactions were performed using unlabeled Forward ACR Primer 1 andFAM-labeled Forward ACR Primer 2. ssDNA oligo template was added to thereactions at a 33-fold molar excess. Reactions were set up at roomtemperature and incubated at 35° C. for 20 min., and then thermocycledin a MultiGene Labnet thermocycler. The thermocycling protocol was 95°C. for 5 min., then 40 cycles of 95° C., 30 sec. and 20° C., 1 min. Thereactions were stopped with equal volumes of formamide containing dye,heat denatured, cooled on ice, and load directly onto the denaturinggel. FIG. 20 shows FAM fluorescence of reactions on an unstained 20%acrylamide+urea denaturing gel using the Typhoon Trio+ imaging system.Lane 1 is the no-template control. Lane 2 contains primers pre-heated at35° C. for 20 min. Lane 3 contains primers pre-heated at 95° C. for 15min. ACR primers pretreated for 15 min. at 95° C. show similarreactivity to primers pretreated for 20 min. at 35° C., prior tothermocycling. Also, no decrease in autoligation reactivity was alsoobserved when 95° C.-treated ACR primers were compared to primersincubated at room temperature (data not shown).

Example 19 Determination of Limit of Detection

Reactions were performed using Texas Red-labeled Forward ACR Primer 1,FAM-labeled Forward ACR Primer 2, and unlabeled reverse primers, with atitration of dsDNA oligo template. The reactions were stopped with equalvolumes of formamide containing dye, heat denatured, cooled on ice, andload directly onto the denaturing gel. FIG. 10 shows FAM/Texas Red FRETfluorescence of reactions on an unstained 20% acrylamide+urea denaturinggel using the Typhoon Trio+ imaging system with excitation channel 488nm and emission channel 610 nm. Lane 3 is from 10,000 molecules, Lane 4is from 1,000 molecules, and lane 5 is from 40 molecules of template.The band in the middle of the gel is observed in both the loading dyelane (Lane 1) and the lane with only template (Lane 2). Autoligationproducts are visible from reactions containing 10,000 and 1,000molecules, but not from the reaction containing 40 molecules. Theautoligation product is also not observed without template (data notshown). These results demonstrate the feasibility of exponentialamplification using ACR primers.

The invention claimed is:
 1. A reagent composition for amplifying atarget nucleic acid sequence comprising: a first forward oligonucleotidehaving a first bond-forming reactive moiety; a second forwardoligonucleotide having a second bond-forming reactive moiety; a firstreverse oligonucleotide having a third bond-forming reactive moiety; anda second reverse oligonucleotide having a fourth bond-forming reactivemoiety; wherein the first forward oligonucleotide, the second forwardoligonucleotide, the first reverse oligonucleotide, or the secondreverse oligonucleotide is configured to specifically anneal with thetarget nucleic acid sequence; and wherein the first forwardoligonucleotide comprises the first bond-forming reactive moiety that isan azide group conjugated to a 3′ terminus of the first forwardoligonucleotide, and the second forward oligonucleotide comprises thesecond bond-forming reactive moiety that is an alkyne group conjugatedto a 5′ terminus of the second forward oligonucleotide; or the firstforward oligonucleotide comprises the first bond-forming reactive moietythat is an alkyne group conjugated to a 3′ terminus of the first forwardoligonucleotide, and the second forward oligonucleotide comprises thesecond bond-forming reactive moiety that is an azide group conjugated toa 5′ terminus of the second forward oligonucleotide; the first reverseoligonucleotide comprises the third bond-forming reactive moiety that isan azide group conjugated to a 3′ terminus of the first reverseoligonucleotide, and the second reverse oligonucleotide comprises thefourth bond-forming reactive moiety that is an alkyne group conjugatedto a 5′ terminus of the second reverse oligonucleotide; or the firstreverse oligonucleotide comprises the third bond-forming reactive moietythat is an alkyne group conjugated to a 3′ terminus of the first reverseoligonucleotide, and the second reverse oligonucleotide comprises thefourth bond-forming reactive moiety that is an azide group conjugated toa 5′ terminus of the second reverse oligonucleotide; the first forwardoligonucleotide and the second forward oligonucleotide are fullycomplementary to the target nucleic acid sequence and the firstbond-forming reactive moiety and the second bond-forming reactive moietyare juxtaposed when annealed to the target nucleic acid sequence; andthe first reverse oligonucleotide and the second reverse oligonucleotideare fully complementary to a complement of the target nucleic acidsequence and the third bond-forming reactive moiety and the fourthbond-forming reactive moiety are juxtaposed when annealed to thecomplement of the target nucleic acid sequence.
 2. The reagentcomposition of claim 1, wherein the first bond-forming reactive moietyor the third bond-forming reactive moiety is an azide, and the secondbond-forming reactive moiety or the fourth bond-forming reactive moietyis an alkyne.
 3. The reagent composition of claim 1, wherein the firstbond-forming reactive moiety or the third bond-forming reactive moietyis an alkyne, and the second bond-forming reactive moiety or the fourthbond-forming reactive moiety is an azide.
 4. The reagent composition ofclaim 2, wherein the alkyne is cycloctyne.
 5. The reagent composition ofclaim 1, wherein the first bond-forming reactive moiety and the secondbond-forming reactive moiety are configured to form a chemical bond inan absence of an enzyme when juxtaposed on the target nucleic acidsequence.
 6. The reagent composition of claim 1, wherein the thirdbond-forming reactive moiety and the fourth bond-forming reactive moietyare configured to form a chemical bond in an absence of an enzyme whenjuxtaposed on the target nucleic acid sequence.
 7. The reagentcomposition of claim 1, wherein at least one of the first forwardoligonucleotide, the second forward oligonucleotide, the first reverseoligonucleotide, or the second reverse oligonucleotide is conjugated toa detectable group.
 8. The reagent composition of claim 1, wherein thefirst forward oligonucleotide or the first reverse oligonucleotidecomprises a FRET donor fluorophore and the second forwardoligonucleotide or the second or reverse oligonucleotide comprises aFRET acceptor fluorophore.
 9. The reagent composition of claim 1,wherein the first forward oligonucleotide or the first reverseoligonucleotide comprises a FRET acceptor fluorophore and the secondforward oligonucleotide or the second reverse oligonucleotide comprisesa FRET donor fluorophore.
 10. The reagent composition of claim 1,wherein the first forward oligonucleotide or the first reverseoligonucleotide comprises a quenched dye or detectable group and thesecond forward oligonucleotide or the second reverse oligonucleotidecomprises a quenching moiety.
 11. The reagent composition of claim 1,wherein the first forward oligonucleotide or the first reverseoligonucleotide comprises a quenching moiety and the second forwardoligonucleotide or the second reverse oligonucleotide comprises aquenched dye or detectable group.
 12. The reagent composition of claim1, further comprising a double-stranded nucleic acid binding dye. 13.The reagent composition of claim 1, wherein each of the first forwardoligonucleotide, the second forward oligonucleotide, the first reverseoligonucleotide, and the second reverse oligonucleotide is from 5 to 35nucleotides in length.
 14. The reagent composition of claim 1, furthercomprising a buffer.
 15. The reagent composition of claim 1, furthercomprising a salt.
 16. The reagent composition of claim 1, wherein thereagent composition does not comprise an enzyme.
 17. The reagentcomposition of claim 1, wherein the reagent composition does notcomprise a nucleotide.
 18. A kit for amplifying two or more targetnucleic acid sequences, comprising: a first forward oligonucleotidehaving a first bond-forming reactive moiety; a second forwardoligonucleotide having a second bond-forming reactive moiety; a firstreverse oligonucleotide having a third bond-forming reactive moiety; anda second reverse oligonucleotide having a fourth bond-forming reactivemoiety; wherein the first forward oligonucleotide, the second forwardoligonucleotide, the first reverse oligonucleotide, or the secondreverse oligonucleotide is configured to specifically anneal with atarget nucleic acid sequence; and wherein the first forwardoligonucleotide comprises the first bond-forming reactive moiety that isan azide group conjugated to a 3′ terminus of the first forwardoligonucleotide, and the second forward oligonucleotide comprises thesecond bond-forming reactive moiety that is an alkyne group conjugatedto a 5′ terminus of the second forward oligonucleotide; or the firstforward oligonucleotide comprises the first bond-forming reactive moietythat is an alkyne group conjugated to a 3′ terminus of the first forwardoligonucleotide, and the second forward oligonucleotide comprises thesecond bond-forming reactive moiety that is an azide group conjugated toa 5′ terminus of the second forward oligonucleotide; the first reverseoligonucleotide comprises the third bond-forming reactive moiety that isan azide group conjugated to a 3′ terminus of the first reverseoligonucleotide, and the second reverse oligonucleotide comprises thefourth bond-forming reactive moiety that is an alkyne group conjugatedto a 5′ terminus of the second reverse oligonucleotide; or the firstreverse oligonucleotide comprises the third bond-forming reactive moietythat is an alkyne group conjugated to a 3′ terminus of the first reverseoligonucleotide, and the second reverse oligonucleotide comprises thefourth bond-forming reactive moiety that is an azide group conjugated toa 5′ terminus of the second reverse oligonucleotide; the first forwardoligonucleotide and the second forward oligonucleotide are fullycomplementary to the target nucleic acid sequence and the firstbond-forming reactive moiety and the second bond-forming reactive moietyare juxtaposed when annealed to the target nucleic acid sequence; andthe first reverse oligonucleotide and the second reverse oligonucleotideare fully complementary to a complement of the target nucleic acidsequence and the third bond-forming reactive moiety and the fourthbond-forming reactive moiety are juxtaposed when annealed to thecomplement of the target nucleic acid sequence.
 19. The kit of claim 18,wherein the two or more target nucleic acid sequences comprise a firsttarget nucleic acid sequence and a second target nucleic acid sequencesuch that the second target nucleic acid sequence differs from the firsttarget nucleic acid sequence by at least a single nucleotide or anucleotide base pair.
 20. The kit of claim 19, wherein the second targetnucleic acid sequence differs from the first target nucleic acidsequence by the single nucleotide or the nucleotide base pair.
 21. Thekit of claim 18, further including a reagent that disrupts a DNA duplex.22. The kit of claim 21, wherein the reagent is an organic solvent, ahigh pH solution, a cross-linking reagent, a chaotropic agent, adisulfide bond reducer, an oligo wedge, or a low salt concentrationsolution.
 23. The kit of claim 18, wherein the first bond-formingreactive moiety or the third bond-forming reactive moiety is an azide,and the second bond-forming reactive moiety or the fourth bond-formingreactive moiety is an alkyne.
 24. The kit of claim 18, wherein the firstbond-forming reactive moiety or the third bond-forming reactive moietyis an alkyne, and the second bond-forming reactive moiety or the fourthbond-forming reactive moiety is an azide.
 25. The kit of claim 23,wherein the alkyne is cycloctyne.
 26. The kit of claim 18, wherein thefirst bond-forming reactive moiety and the second bond-forming reactivemoiety are configured to form a chemical bond in an absence of an enzymewhen juxtaposed on the target nucleic acid sequence.
 27. The kit ofclaim 18, wherein the third bond-forming reactive moiety and the fourthbond-forming reactive moiety are configured to form a chemical bond inan absence of an enzyme when juxtaposed on the target nucleic acidsequence.
 28. The kit of claim 18, wherein at least one of the firstforward oligonucleotide, the second forward oligonucleotide, the firstreverse oligonucleotide, or the second reverse oligonucleotide isconjugated to a detectable group.
 29. The kit of claim 18, wherein thefirst forward oligonucleotide or the first reverse oligonucleotidecomprises a FRET donor fluorophore and the second forwardoligonucleotide or the second reverse oligonucleotide comprises a FRETacceptor fluorophore.
 30. The kit of claim 18, wherein the first forwardoligonucleotide or the first reverse oligonucleotide comprises a FRETacceptor fluorophore and the second forward oligonucleotide or thesecond reverse oligonucleotide comprises a FRET donor fluorophore. 31.The kit of claim 18, wherein the first forward oligonucleotide or thefirst reverse oligonucleotide comprises a quenched dye or detectablegroup and the second forward oligonucleotide or the second reverseoligonucleotide comprises a quenching moiety.
 32. The kit of claim 18,wherein the first forward oligonucleotide or the first reverseoligonucleotide comprises a quenching moiety and the second forwardoligonucleotide or the second reverse oligonucleotide comprises aquenched dye or detectable group.
 33. The kit of claim 18, furthercomprising a double-stranded nucleic acid binding dye.
 34. The kit ofclaim 18, wherein each of the first forward oligonucleotide, the secondforward oligonucleotide, the first reverse oligonucleotide, or thesecond reverse oligonucleotide is from 5 to 35 nucleotides in length.35. The kit of claim 18, further comprising a buffer.
 36. The kit ofclaim 18, further comprising a salt.
 37. The kit of claim 18, whereinthe kit does not comprise an enzyme.
 38. The kit of claim 18, whereinthe kit does not comprise a nucleotide.
 39. The composition of claim 3,wherein the alkyne is cycloctyne.
 40. The kit of claim 24, wherein thealkyne is cycloctyne.